The present technology relates to a method of detecting nucleic acids, a method of optically observing a sample and a fluorescent substance. More particularly, the present technology relates to a method of detecting nucleic acids, a method of optically observing a sample, both method being based on fluorescence emitted from the nucleic acids contacted with copper, and a fluorescent substance including copper and nucleic acids.
In recent years, technical research has been widely taken place by using nucleic acids in a variety of fields including a medical field, a drug discovery field, a clinical examination field, a food field, an agricultural field, an engineering field, a forensic medicine field and a criminal identification field. Recently, a lab-on-chip is technically developed and is practically used to stain, detect, amplify etc. nucleic acids within micro-scale flow channels disposed in a microchip.
As a fundamental technique for detecting nucleic acids, there is a method of using a fluorescent pigment for staining the nucleic acids. Many fluorescent pigments are known such as hoechst33342, DAPI, ethidium bromide, SYBR green and the like. For example, hoechst33342 and DAPI are used for the purpose of staining nucleic acids in cells or tissues in a flow cytometry or a microscope. The ethidium bromide is frequently used to stain nucleic acid molecules in an electrophoresis. The SYBR green and the like are also used for the purpose of detecting in a real time an amplification process of the nucleic acids in a nucleic acid amplification technology such as a polymerase chain reaction.
In relation to the present technology, conventionally known autofluorescence shown by cells upon a fluorescence observation will be described. One of the fluorescence is orange-colored autofluorescence shown by UV-irradiated cells in the presence of copper. For example, it is reported that cells of a particular part of a drosophila larvae midgut emits orange-colored fluorescence when copper is added (see Non-Patent Documents 1 to 8). The cells where the orange-colored fluorescence is especially strongly observed in the drosophila larvae midgut are called as “copper cells” or the like. It is reported that the fluorescence is observed at cells around the copper cells (Non-Patent Document 4) and an entire body wall of the larvae (Non-Patent Document 2) when the concentration of the copper added is increased.
There is a description that the above-mentioned orange-colored fluorescence is observed at both cytoplasms and cell nuclei in cells, and, in particular, is detected predominantly in grains of the cytoplasms (see Non-Patent Documents 2 to 4 and 7). There is a description that a wavelength range of fluorescence is 590 to 630 nm, a peak wavelength is 610 nm and a maximum excitation wavelength is 340 nm (see Non-Patent Document 3).
Also for organisms other than drosophila, autofluorescence having similar properties is observed. For example, there is reported that orange-colored fluorescence (having a peak wavelength of 605 nm) is observed in an individual liver to which copper is added by UV excitation (excitation wavelength of 310 nm) in rat experiments (see Non-Patent Document 9). Furthermore, there is reported that similar fluorescence is observed in a kidney of a model rat having a kidney and a liver where copper is accumulated with aging (see Non-Patent Document 10). Also, the autofluorescence having similar properties is reported in yeast (see Non-Patent Document 11) and human liver tissues of a Wilson's disease patient (see Non-Patent Document 12). The Wilson's disease is a genetic disorder of insufficient excretion of copper and accumulation of copper in liver cells.
As the above-described fluorescent substance emitting orange-colored fluorescence, a composite of copper and metallothionein (MT) (hereinafter abbreviated to as “Cu-MT”) is presumed (see Non-Patent Documents 14 to 23). The Cu-MT has wavelength properties such as an excitation wavelength of 305 nm and a fluorescence wavelength of 565 nm in Non-Patent Document 13, and an excitation wavelength of 310 nm and a fluorescence wavelength of 570 nm in Non-Patent Document 17. It is conceivable that the Cu-MT contain monovalent copper ions (Cu(I)) (see Non-Patent Documents 13, 15, 17, 19 and 23).
As the fluorescent substance containing copper, a compound containing pyrimidine or mercaptide that emits fluorescence by interacting pyrimidine or mercaptide with copper is widely known (see Non-Patent Documents 24 to 29).
On the other hand, an interaction of various metal ions with nucleic acids has been traditionally studied. For example, it is known that when monovalent copper ions are interacted with nucleic acids, a minor amount of copper contained in cell nuclei stabilizes a nucleic acid structure, but hurts DNAs under coexistence of hydrogen peroxide (see Non-Patent Document 30). It is also reported that an interaction with copper changes an absorption spectrum of DNAs (see Non-Patent Documents 30 and 31). Further, it is reported that the change in the absorption spectrum depends on base sequences (specifically, a polymer having a G-C pair and a polymer having an A-T pair) of the DNAs (see Non-Patent Document 30).